Similar localization of conformational IgE epitopes on the house dust mite allergens Der p 5 and Der p 21 despite limited IgE cross-reactivity.

BACKGROUND: Due to high IgE recognition frequency and high allergenic activity, Der p 5 and Der p 21 are clinically important house dust mite (HDM) allergens. The objective of this study was to characterize the immunodominant IgE epitopes of Der p 5 and Der p 21 responsible for their high allergenic activity. METHODS: A panel of 12 overlapping peptides spanning the Der p 5 and Der p 21 sequence were synthesized to search for sequential IgE epitopes by direct testing for allergic patients' IgE reactivity. Peptide-specific antibodies raised in rabbits were used in inhibition studies for localizing conformational IgE epitopes which were visualized on the surfaces of the allergen structures by molecular modelling. IgE cross-reactivity between the allergens was investigated by IgE inhibition studies. RESULTS: Immunodominant IgE epitopes defined by allergic patients' IgE on Der p 5 and Der p 21 were primarily of the conformational, discontinuous type including N- and C-terminal portions of the protein. They could be located on each allergen on one area with similar localization, but despite similar structure of the allergens, no relevant IgE cross-reactivity could be detected. CONCLUSION: Our study shows that Der p 5 and Der p 21 contain a major conformational IgE epitope-containing area located on similar portions of their structure, but they lack relevant IgE cross-reactivity. These data are important for the development of modern allergy vaccines based on defined molecules for allergen-specific immunotherapy of HDM allergy.

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. METHODS: A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. RESULTS: A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. CONCLUSION: Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients.

BTK inhibition is a potent approach to block IgE-mediated histamine release in human basophils.

BACKGROUND: Recent data suggest that Bruton's tyrosine kinase (BTK) is an emerging therapeutic target in IgE receptor (IgER)-cross-linked basophils. METHODS: We examined the effects of four BTK inhibitors (ibrutinib, dasatinib, AVL-292, and CNX-774) on IgE-dependent activation and histamine release in blood basophils obtained from allergic patients (n=11) and nonallergic donors (n=5). In addition, we examined the effects of these drugs on the growth of the human basophil cell line KU812 and the human mast cell line HMC-1. RESULTS: All four BTK blockers were found to inhibit anti-IgE-induced histamine release from basophils in nonallergic subjects and allergen-induced histamine liberation from basophils in allergic donors. Drug effects on allergen-induced histamine release were dose dependent, with IC50 values ranging between 0.001 and 0.5 µmol/L, and the following rank order of potency: ibrutinib>AVL-292>dasatinib>CNX-774. The basophil-targeting effect of ibrutinib was confirmed by demonstrating that IgE-dependent histamine release in ex vivo blood basophils is largely suppressed in a leukemia patient treated with ibrutinib. Dasatinib and ibrutinib were also found to counteract anti-IgE-induced and allergen-induced upregulation of CD13, CD63, CD164, and CD203c on basophils, whereas AVL-292 and CNX-774 showed no significant effects. Whereas dasatinib and CNX-774 were found to inhibit the growth of HMC-1 cells and KU812 cells, no substantial effects were seen with ibrutinib or AVL-292. CONCLUSIONS: BTK-targeting drugs are potent inhibitors of IgE-dependent histamine release in human basophils. The clinical value of BTK inhibition in the context of allergic diseases remains to be determined.

Pru p 3, a marker allergen for lipid transfer protein sensitization also in Central Europe.

In the Mediterranean area, lipid transfer proteins (LTPs) are important causes of plant-food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extract-based diagnosis is often complicated by co-sensitization to Bet v 1, the major birch pollen allergen, its cross-reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant-derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant-food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients.

Infant milk formulas differ regarding their allergenic activity and induction of T-cell and cytokine responses.

BACKGROUND: Several hydrolyzed cow's milk (CM) formulas are available for avoidance of allergic reactions in CM-allergic children and for prevention of allergy development in high-risk infants. Our aim was to compare CM formulas regarding the presence of immunoreactive CM components, IgE reactivity, allergenic activity, ability to induce T-cell proliferation, and cytokine secretion. METHODS: A blinded analysis of eight CM formulas, one nonhydrolyzed, two partially hydrolyzed (PH), four extensively hydrolyzed (EH), and one amino acid formula, using biochemical techniques and specific antibody probes was conducted. IgE reactivity and allergenic activity of the formulas were tested with sera from CM-allergic patients (n = 26) in RAST-based assays and with rat basophils transfected with the human FcεRI, respectively. The induction of T-cell proliferation and the secretion of cytokines in Peripheral blood mononuclear cell (PBMC) culture from CM allergic patients and nonallergic individuals were assessed. RESULTS: Immune-reactive α-lactalbumin and ß-lactoglobulin were found in the two PH formulas and casein components in one of the EH formulas. One PH formula and the EH formula containing casein components showed remaining IgE reactivity, whereas the other hydrolyzed formulas lacked IgE reactivity. Only two EH formulas and the amino acid formula did not induce T-cell proliferation and proinflammatory cytokine release. The remaining formulas varied regarding the induction of Th2, Th1, and proinflammatory cytokines. CONCLUSION: Our results show that certain CM formulas without allergenic and low proinflammatory properties can be identified and they may also explain different outcomes obtained in clinical studies using CM formulas.

Epitope specificity determines cross-protection of a SIT-induced IgG4 antibody.

BACKGROUND: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT). METHODS: We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments. RESULTS: Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity. CONCLUSIONS: Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.

Natural clinical tolerance to peanut in African patients is caused by poor allergenic activity of peanut IgE.

BACKGROUND: In Africa, peanuts are frequently consumed, but severe allergic reactions are rare. We investigated immunological patterns of clinical tolerance to peanut in peanut-sensitized but asymptomatic patients from central Africa compared to peanut-allergic and peanut-sensitized but asymptomatic patients from Sweden. METHODS: Sera from allergic patients (n = 54) from Zimbabwe sensitized to peanut but without allergic symptoms to peanut, and sera from peanut-allergic (n = 25) and peanut-sensitized but asymptomatic (n = 25) patients from Sweden were analyzed toward peanut allergen components (Ara h 1-3, 6, 8-9) and other allergen molecules from important allergen sources using microarray. IgE to Ara h 2 peptide epitopes was analyzed, and allergenic activity was assessed by basophil activation assay. RESULTS: Forty-six percent of the African and all peanut-allergic Swedish patients showed IgE toward one of the highly allergenic peanut allergens (Ara h 1-3, 6, 9). However, 48% of the African patients had IgE to cross-reactive carbohydrate determinants (CCDs) with low allergenic activity and 60% of the Swedish asymptomatic patients had IgE against the PR protein Ara h 8. IgG and IgG4 specificities and levels could not discriminate between the African asymptomatic and Swedish peanut-allergic patients. Asymptomatic patients almost lacked IgE to Ara h 2 peptides, which were recognized by peanut-allergic patients. Peanut IgE from peanut asymptomatic patients showed poor allergenic activity compared with IgE from peanut-allergic patients. CONCLUSIONS: Natural clinical tolerance to peanut in the African patients can be caused by IgE to low allergenic peanut components and by poor allergenic activity of peanut-specific IgE.

Multiple independent IgE epitopes on the highly allergenic grass pollen allergen Phl p 5.

BACKGROUND: Group 5 allergens are small proteins that consist of two domains. They belong to the most potent respiratory allergens. OBJECTIVE: To determine the binding sites and to study allergic patients' IgE recognition of the group 5 allergen (Phl p 5) from timothy grass pollen using human monoclonal IgE antibodies that have been isolated from grass pollen allergic patients. METHODS: Using recombinant isoallergens, fragments, mutants and synthetic peptides of Phl p 5, as well as peptide-specific antibodies, the interaction of recombinant human monoclonal IgE and Phl p 5 was studied using direct binding and blocking assays. Cross-reactivity of monoclonal IgE with group 5 allergens in several grasses was studied and inhibition experiments with patients' polyclonal IgE were performed. RESULTS: Monoclonal human IgE showed extensive cross-reactivity with group 5 allergens in several grasses. Despite its small size of 29 kDa, four independent epitope clusters on isoallergen Phl p 5.0101, two in each domain, were recognized by human IgE. Isoallergen Phl p 5.0201 carried two of these epitopes. Inhibition studies with allergic patients' polyclonal IgE suggest the presence of additional IgE epitopes on Phl p 5. CONCLUSIONS & CLINICAL RELEVANCE: Our results reveal the presence of a large number of independent IgE epitopes on the Phl p 5 allergen explaining the high allergenic activity of this protein and its ability to induce severe allergic symptoms. High-density IgE recognition may be a general feature of many potent allergens and form a basis for the development of improved diagnostic and therapeutic procedures in allergic disease.

Hypoallergenic derivatives of Fel d 1 obtained by rational reassembly for allergy vaccination and tolerance induction.

BACKGROUND AND OBJECTIVE: The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was to engineer recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction. METHODS: Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen-specific blocking IgG antibodies and T cell epitopes. Mosaic proteins were expressed in Escherichia coli using codon-optimized synthetic genes and compared with Fel d 1 regarding structural fold by circular dichroism, IgE-binding capacity, activation of allergic patients' basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization. RESULTS: Although each of the mosaic proteins had lost the alpha-helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two (Fel d 1 MF) in which the cysteines of Fel d 1 MC were replaced by serines. Immunization of rabbits with Fel d 1 MB, MC and MF induced high levels of IgG antibodies that inhibited IgE reactivity of cat-allergic patients to Fel d 1 in a comparable manner as IgG induced with the wild-type allergen. CONCLUSIONS: We report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat-allergic patients.

Prophylactic and therapeutic vaccination with carrier-bound Bet v 1 peptides lacking allergen-specific T cell epitopes reduces Bet v 1-specific T cell responses via blocking antibodies in a murine model for birch pollen allergy.

BACKGROUND: Vaccines consisting of allergen-derived peptides lacking IgE reactivity and allergen-specific T cell epitopes bound to allergen-unrelated carrier molecules have been suggested as candidates for allergen-specific immunotherapy. OBJECTIVE: To study whether prophylactic and therapeutic vaccination with carrier-bound peptides from the major birch pollen allergen Bet v 1 lacking allergen-specific T cell epitopes has influence on Bet v 1-specific T cell responses. METHODS: Three Bet v 1-derived peptides, devoid of Bet v 1-specific T cell epitopes, were coupled to KLH and adsorbed to aluminium hydroxide to obtain a Bet v 1-specific allergy vaccine. Groups of BALB/c mice were immunized with the peptide vaccine before or after sensitization to Bet v 1. Bet v 1- and peptide-specific antibody responses were analysed by ELISA. T cell and cytokine responses to Bet v 1, KLH, and the peptides were studied in proliferation assays. The effects of peptide-specific and allergen-specific antibodies on T cell responses and allergic lung inflammation were studied using specific antibodies. RESULTS: Prophylactic and therapeutic vaccination with carrier-bound Bet v 1 peptides induced a Bet v 1-specific IgG antibody response without priming/boosting of Bet v 1-specific T cells. Prophylactic and therapeutic vaccination of mice with the peptide vaccine induced Bet v 1-specific antibodies which suppressed Bet v 1-specific T cell responses and allergic lung inflammation. CONCLUSION AND CLINICAL RELEVANCE: Vaccination with carrier-bound allergen-derived peptides lacking allergen-specific T cell epitopes induces allergen-specific IgG antibodies which suppress allergen-specific T cell responses and allergic lung inflammation.

Carrier-bound nonallergenic Der p 2 peptides induce IgG antibodies blocking allergen-induced basophil activation in allergic patients.

BACKGROUND: More than 90% of house dust mite-allergic patients are sensitized to the major Dermatophagoides pteronyssinus allergen, Der p 2. The aim of this study was to develop and characterize an allergy vaccine based on carrier-bound Der p 2 peptides, which should allow reducing IgE- and T-cell-mediated side-effects during specific immunotherapy (SIT). METHODS: Five Der p 2 peptides (P1-P5) were synthesized and analyzed regarding IgE reactivity and allergenic activity. Lymphoproliferative and cytokine responses induced with Der p 2 and Der p 2 peptides were determined in peripheral blood mononuclear cells from mite-allergic patients. Der p 2-specific IgG antibodies induced with carrier-bound Der p 2 peptides in mice and rabbits were tested for their capacity to inhibit IgE binding and basophil activation in allergic patients. RESULTS: Of five overlapping peptides (P1-P5) covering the Der p 2 sequence, two peptides (P2 and P4) were identified, which showed no relevant IgE reactivity, allergenic activity, and induced lower Der p 2-specific T-cell activation than Der p 2. However, when coupled to a carrier, P2 and P4 induced Der p 2-specific IgG antibodies in animals, which inhibited allergic patients' IgE binding to the allergen and allergen-induced basophil activation similar as antibodies induced with Der p 2. CONCLUSIONS: Carrier-bound Der p 2 peptides should allow avoiding IgE-mediated side-effects, and because of their low potential to activate allergen-specific T cells, they may reduce late-phase side-effects during SIT. Further, these peptides may be also useful for prophylactic vaccination.

Immunoglobulin E antibody reactivity to bacterial antigens in atopic dermatitis patients.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 20% children and 9% adults world-wide. AD patients are often sensitized against a broad variety of allergens and more than 90% of them suffer from skin superinfections with Staphylococcus aureus. OBJECTIVE: In this study, we searched for the presence of specific IgE antibodies against S. aureus and Escherichia coli antigens in AD patients. METHODS: Sera from AD patients (n=79), patients suffering only from allergic rhinoconjunctivitis (n=41) or allergic asthma (n=37) were tested for IgE reactivity to nitrocellulose-blotted S. aureus, E. coli and gut bacterial antigens. IgE-reactive bacterial antigens were affinity purified and identified by mass spectrometry. RESULTS: More than 30% of AD patients but not patients suffering only from allergic rhinoconjunctivitis and asthma or non-allergic persons exhibited IgE binding to several protein antigens among them DNA-binding and ribosomal proteins and flagellin. Patients with severe skin manifestations showed more frequently IgE reactivity to S. aureus compared with AD patients with mild symptoms. Positive immediate and late skin test reactions could be induced in sensitized AD patients with S. aureus extract. CONCLUSION AND CLINICAL RELEVANCE: Specific IgE reactivities against a variety of bacterial antigens were observed in a subgroup comprising a third of AD patients and may contribute to allergic inflammation.

Antibodies induced with recombinant VP1 from human rhinovirus exhibit cross-neutralisation.

Human rhinoviruses (HRVs) are the major cause of the common cold and account for 30-50% of all acute respiratory illnesses. Although HRV infections are usually harmless and invade only the upper respiratory tract, several studies demonstrate that HRV is involved in the exacerbation of asthma. VP1 is one of the surface-exposed proteins of the viral capsid that is important for the binding of rhinoviruses to the corresponding receptors on human cells. Here we investigated its potential usefulness for vaccination against the common cold. We expressed VP1 proteins from two distantly related HRV strains, HRV89 and HRV14, in Escherichia coli. Mice and rabbits were immunised with the purified recombinant proteins. The induced antibodies reacted with natural VP1 and with whole virus particles as shown by immunoblotting and immunogold electron microscopy. They exhibited strong cross-neutralising activity for different HRV strains. Therefore, recombinant VP1 may be considered a candidate HRV vaccine to prevent HRV-induced asthma exacerbations.

Genetically engineered and synthetic allergen derivatives: candidates for vaccination against type I allergy.

Type I allergy, a hypersensitivity disease affecting almost 20% of the population worldwide, is based on the IgE recognition of otherwise harmless antigens (i.e., allergens). Allergen-induced crosslink of effector cell-bound IgE antibodies leads to the release of biological mediators and thus to immediate disease symptoms (allergic rhinitis, conjunctivitis and asthma). Specific immunotherapy, the only causative treatment of Type I allergy, is based on the administration of increasing doses of allergens to allergic patients in order to yield allergen-specific non-responsiveness. Major disadvantages are 1. that current forms of allergen immunotherapy are performed with allergens difficult to standardize which cannot be matched to the patients reactivity profile and 2. that the administration of active allergen preparations can cause anaphylactic side effects. Through the application of molecular biological techniques many relevant environmental allergens have been produced as active recombinant proteins which allow component-resolved allergy diagnosis and thus represent the basis for patient-tailored forms of immunotherapy. Here we review molecular strategies which have been recently applied to generate genetically engineered and synthetic hypoallergenic allergen derivatives for patient-tailored and safe vaccination against Type I allergy.